This invention relates to therapeutic immunoconjugates, compositions that can be utilized to make them, and methods of making and using such compositions and immunoconjugates.
The natural products CC-1065 and the duocarmycins are potent cytotoxic agents that bind to the minor groove of DNA. They are characterized by a fused cyclopropyl ring (dotted box in structures below) that is destabilized by conformational changes induced upon binding within the minor groove and reacts with a DNA adenine base in a ring-opening alkylation reaction. The damage to the DNA can be irreparable, leading to cell death.

The potency of these natural products has stimulated research aimed at developing analogs useful as anti-cancer drugs. See, e.g., Cacciari et al. 2000 and Suckling 2004. (Full citations of the documents cited herein by first author or inventor and year are listed at the end of this specification. Such listed documents are incorporated herein by reference.) The analogs have either the fused cyclopropyl pharmacophore or a ring-opened (seco) equivalent thereof, as shown in the structures below, where Ar represents an aromatic ring that typically is phenyl or pyrrole and X represents a leaving group such as Cl or Br. The seco form is convertible to the cyclopropyl form by the elimination of HX, a process that can occur either in vitro or in vivo.

Hereinafter the term “minor groove binding agent” or “MGBA” will be used to refer to CC-1065/duocarmycin type compounds having the fused cyclopropyl ring or its seco form, although other types of DNA minor groove binding compounds are known.
Immunoconjugates represent an area of high current interest in anti-cancer therapy. In an immunoconjugate, a drug moiety is conjugated (covalently linked) to an antibody whose antigen is uniquely expressed or overexpressed by a cancer cell (“tumor associated antigen”). In binding to its antigen, the antibody functions as a targeting agent for delivering the drug moiety to the cancer cell with high specificity. The antigen can be a protein on the surface of the cancer cell. Upon binding of the antibody to the antigen, the antigen-immunoconjugate complex is internalized and eventually finds its way inside a vesicular body such as a lysosome, where the covalent linker between the drug moiety and the antibody is cleaved, liberating the drug moiety to exert its cytotoxic effect. Alternatively, the tumor associated antigen can be one that is secreted by tumor cells into the vicinal extracellular space.
Advantageously, the covalent linker is designed such that cleavage is effected by a factor prevalent inside a lysosome but not in plasma. One such factor is the lower lysosomal pH, so that the covalent linker can be an acid-sensitive group such as a hydrazone. Another such factor is the generally higher intracellular concentration of glutathione, allowing for the cleavage of disulfide covalent linkers by a disulfide exchange mechanism. Yet another such factor is the presence of lysosomal enzymes such as cathepsin B, which can cleave peptidyl linkers designed to be preferred substrates (Dubowchik et al. 2002).
Their potency makes MGBAs attractive candidates for the drug moiety in an immunoconjugate. Illustrative disclosures relating to MGBAs and their use in immunoconjugates include: Boyd et al. 2008 and 2010; Chen et al. 2010; Gangwar et al. 2008; Ng et al. 2002, 2006a, 2006b, 2009a, 2009b, and 2010; and Sufi et al. 2010.
There exists the possibility of adventitious cleavage of the covalent linker while the immunoconjugate is still in general circulation and has not yet been delivered to the target cancer cell, resulting in the premature release of the drug moiety and posing the risk of systemic toxicity. Such risk is of particular concern where the drug moiety is a highly potent cytotoxin such as an MGBA. However, where an immunoconjugate employs a seco-MGBA, the risk can be reduced by derivatizing the phenolic hydroxyl group to block conversion to the cyclopropyl form. Then, in the event of adventitious cleavage, what is released is an inactive derivatized seco-MGBA. By selecting a derivative that is cleaved inside a lysosome, the derivative functions as a prodrugging group and provides a safety factor: two cleavages, of the linker and of the prodrugging group, must occur before active cytotoxin is released.
One such prodrugging group is a carbamate, which can be cleaved by lysosomal and/or cytosolic carboxyesterase as shown below. (Ra and Rb represent generic radical groups.) For illustrative disclosures relating to seco-MGBA immunoconjugates prodrugged with a carbamate group, see Aristoff et al. 1991; Boger et al. 1999; Boyd et al. 2008 and 2010; Chen et al. 2010; Gangwar et al. 2008; Kobayashi 1994; Ng et al. 2002, 2006a, 2006b, 2009a, 2009b, and 2010; Sufi et al. 2010; and Zhao et al. 2010.

Another prodrugging group that can be used with seco-MGBAs is a phosphate, in which case the cleaving enzyme is a phosphatase, found inside lysosomes and/or in the cytosol. Illustrative disclosures relating to phosphate prodrugging groups in seco-MGBAs include Boyd et al. 2010, Chen et al. 2009, Glazier 2003, King et al. 2011, Kutyavin et al. 1997, Ng et al. 2002, Zhao et al. 2002a and 2002b, and Zhao et al. 2010.
An MGBA compound that has been extensively studied has the structure (A) (Boyd et al. 2008 and Sufi et al. 2010). It has a carbamate prodrugging group; a valine-citrulline (Val-Cit, recited in N-to-C direction, i.e., from the amino (NH2) terminus to the carboxyl (CO2H) terminus) dipeptide linker, designed to be cleaved by cathepsin B (Dubowchik et al. 2002); and a maleimide group, for immunoconjugation by the Michael addition of an antibody sulfhydryl group. An immunoconjugate of compound (A) and an anti-CD70 antibody has been undergoing clinical trials.
